1. Field of the Invention
This invention relates to diagnostic assays and in particular to diagnostic assays which employ phospholipids as assay reagents.
2. Description of the Prior Art
A variety of diagnostic assays are known which include one or more phospholipids as assay reagents. For example, various blood coagulation tests, such as, complete and partial thromboplastin times, prothrombin times, and the like, employ brain and other tissue extracts which include lipids. Similarly, the VDRL (Venereal Disease Research Laboratory) test for syphilis is based on the use of an antigen solution which includes cardiolipin, cholesterol and lecithin.
As with many assay systems, the foregoing assays suffer from the problem of false positives, i.e., for certain patients, the coagulation tests give results indicative of a coagulation problem, when, in fact, the patient's clotting mechanisms are normal, or, in the case of the VDRL test, the patient appears to have syphilis, when in fact he or she is syphilis free.
Prior studies have established a correlation between these false positives and certain diseases. For example, blood samples from patients having the autoimmune disease, systemic lupus erythematosus (SLE), often have prolonged coagulation times, even though clinically, the patients do not exhibit bleeding tendencies and, indeed, in some cases, may suffer from thrombotic episodes. The blood of such patient is said to certain coagulation inhibitors," "lupus inhibitors," "circulating anticoagulants," or "lupus anticoagulants." See T. Exner, et al., "Studies on Phospholipids in the Action of a Lupus Coagulation Inhibitor," Pathology, Vol. 7, 1975, pages 319-328; and P. Thiagarajan, et al., "Monoclonal Immunoglobulin M.lambda. Coagulation Inhibitor with Phospholipid Specificity," J. Clin. Invest., Vol. 66, September 1980, pages 397-405.
It is presently believed that these "inhibitors" are in fact antibodies against phospholipids which are produced by the immune system of patients suffering from SLE. See P. Thiagarajan, et al., supra. Similar anti-phospholipid antibodies have been found in the sera of patients suffering from other autoimmune diseases, such as, connective tissue diseases, Hashimoto's thyroiditis, rheumatoid arthritis, and the like. See P. F. Sparling, "Diagnosis and Treatment of Syphilis," New England Journal of Medicine, Vol. 284, pages 642-653 (1971). Accordingly, patients with these diseases are also likely to give false positives when subjected to lipid-dependent diagnostic assays.
Efforts have been made in the past to solve the problem of false positives in lipid-dependent assays, and, in particular, lipid-dependent coagulation assays, but with only limited success. Thus, Exner, et al., supra, reported that the effect of lupus inhibitor on the Russell viper venom coagulation test could be partially corrected by adding to the reagent mixture what Exner referred to as "partially characterized" phospholipids obtained from bovine cephalin using the Folch procedure. See J. Folch, "Brain Cephalin, A Mixture of Phosphatides. Separation from it of Phosphatidyl Serine, Phosphatidyl Etanolamine, and a Fraction Containing an Inositol Phosphatide," J. Biol. Chem., Vol. 146, 1942, pages 35-41.
Exner tested three phospholipid fractions identified as phosphatidyl ethanolamine, phosphatidyl serine, and inositol phosphatide. As reported by Exner, at low concentrations, each fraction reduced somewhat the clotting times of plasma samples containing lupus inhibitor, but not to the levels observed for normal samples (see page 324 and FIGS. 2A, 2B, and 2C of Exner, et al.). At higher concentrations, the addition of these phospholipid fractions unfortunately changed both the clotting times of the inhibitor-containing samples and the clotting times of the normal samples, i.e., rather than solving the false positive problem, the addition of these phospholipids to the reagent mixture resulted in a change in the overall response, including the baseline, of the assay. Of the three phospholipid fractions tested, Exner stated that the phosphatidyl ethanolamine fraction appeared to give the best corrective effect.
In addition to the Exner work, Thiagarajan, et al., supra, studied the effects on coagulation assays of purified IgM.lambda. paraprotein obtained from a patient whose response to lipid-dependent coagulation tests indicated the presence in the patient's blood of a lupus-type anticoagulant. The purified paraprotein, when added to normal plasma, was found to reproduce the abnormal coagulation time observed with the patient's plasma. Studies using the paraprotein indicated that it reacted with phosphatidylserine and, to a lesser extent, with phosphatidylinositol and phosphatidic acid, but that it did not react with phosphatidylcholine or phosphatidylethanolamine.
A comparison of the results reported by Thiagarajan with those reported by Exner highlights the confusing state of the prior art. Whereas Thiagarajan, et al., found that their lupus anticoagulant would not react with phosphatidylethanolamine, Exner, et al., found just the opposite. Moreover, in Exner's hands, phosphatidylethanolamine distorted the basic character of the assay as evidenced by the fact that the presence of 0.05% phosphatidylethanolamine in the reagent mixture resulted in an over 40% increase in the clotting time of normal plasma and only a 30% decrease in the clotting time of a mixture of 90% normal plasma and 10% patient plasma (see FIG. 2A of Exner, et al.). It is against this background that the present invention was made.